BIOL 4050U Lecture Notes - Lecture 1: Recognition Sequence, Glutamine, Restriction Site
Angiotensin I-converting Enzyme Mutation (Trp1197Stop) causes a Dramatic
increase in Blood ACE
Angiotensin-converting enzyme (ACE) is a central component of the renin-angiotensin system
(RAS) which controls blood pressure by regulating the volume of fluids in the body. It converts
the hormones angiotensin I to active vasoconstrictor angiotensin II.
levels of ACE may lead to risks: cardiovascular or respiratory diseases, including asthma
Pro119Leu substitution enhances the cleavage section process
Codon TGG coding for Trp1197 was substituted in one allele for TGA (stop codon)
Trp1197Stop mutant is associated with Sarcoidosis and Gaucher diseases
Angiotensin I-converting enzyme (ACE, CD143) is a carboxydipeptidase (a protease enzyme
which cleaves a peptide bond at the C-terminus) which plays a key role in regulating blood
pressure
10 volunteers without sarcoidosis was chosen as a control
N1 has a higher than normal ACE activity
Average range is
Blood taken from adults and saliva from children
ACE Activity Assay
Fluorimeteric assay with two ACE assay (5Mm Hip-His-Leu or 2Mm Z-Phe-His-Leu)
20-40 aliquots of plasma diluted in in PBS-BSA () and added to 200 ACE substrate and incubated
at 37, His-Leu product was quantified
Plate Immunoprecipitation Assay
96-well plates coated with ant-ACE mAbs via goat anti-mouse IgG and incubated with plasma
Activity was measured by adding ACE (Hip-His-Leu) directly into well
Sequencing and Genotyping
Two individuals N1 and Dr (who has Pro1199Leu mutation) used as positive control
Then 292 bp DNA fragment amplified by PCR with primer Ex25_292Fw (exon 25) and
Ex25_292Rv (exon 26) using DNA from subjects N1, N2(father of N1), N3(mother of N1) & DR
The restriction endonuclease BsaHI (AcyI) cuts 292 bp PCR with wild type ACE(N3) into two
fragments 235 bp and 57 bp
Two mutations G-to-A in position 3699 (Trp1197Stop)
C-to-T mutation in 3705 (Pro1199Leu)
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Document Summary
Angiotensin i-converting enzyme mutation (trp1197stop) causes a dramatic increase in blood ace. Angiotensin-converting enzyme (ace) is a central component of the renin-angiotensin system (ras) which controls blood pressure by regulating the volume of fluids in the body. It converts the hormones angiotensin i to active vasoconstrictor angiotensin ii. levels of ace may lead to risks: cardiovascular or respiratory diseases, including asthma. Codon tgg coding for trp1197 was substituted in one allele for tga (stop codon) Trp1197stop mutant is associated with sarcoidosis and gaucher diseases. Angiotensin i-converting enzyme (ace, cd143) is a carboxydipeptidase (a protease enzyme which cleaves a peptide bond at the c-terminus) which plays a key role in regulating blood pressure. 10 volunteers without sarcoidosis was chosen as a control. N1 has a higher than normal ace activity. Blood taken from adults and saliva from children. Fluorimeteric assay with two ace assay (5mm hip-his-leu or 2mm z-phe-his-leu)
